The ultimate goal of this proposal is to develop methods that can be applied to produce grafts of cultured human epidermal cells which will provide optimal coverage and rapid healing of burn wounds. In order to develop procedures for expanding our ability to provide such cultured epidermis, we have characterized, in detail, the kinetics of growth and differentiation which occur in cultures of human keratinocytes grown according to a culture method which we developed (1,2). The results of these studies increased our ability to establish cultured grafts which can now be used routinely to promote the rapid healing of burn wounds (1,3,4). We propose to develop the means of increasing the supply of cultured epidermal cell grafts through cryogenic "banking" of cell sheets. We will also test the hypothesis that cell cultures which have been induced to form a stratum corneum in vitro prior to transplantation can provide more rapid coverage and healing of burn wounds than less differentiated cell sheets grown according to our standard cultures methods. The specific aims are: I. To quantitate the effects of cryopreservation on: A. The growth and maturation kinetics of keratinocyte suspensions. B. The ability of the cultured cell sheets to function as transplants in vivo. II. To develop methods to promote the terminal differentiation of epidermal cells in culture. Our efforts will focus on the utilization of chemical and/or non-toxic biological agents which can be used as medium supplements. Preliminary experiments indicate that sodium-N-butyrate (NaB) can enhance terminal differentiation of keratinocytes when added to culture for a short period of time. These studies will be expanded to: A. Characterize the cell subpopulation and growth kinetics of keratinocytes induced to form enucleated corneocytes. B. Quantitate cytoskeletal protein expression and cellular organization during in vitro differentiation to enucleated corneocytes. C. Examine the ability of cultured cell sheets which contain a stratum corneum cell layer to function as transplants in vivo.